This proposal describes experiments to investigate processing of pre-messenger RNA in higher eucaryotes. In the last several years, our understanding of the mechanics of pre-messenger RNA processing has increased considerably, in no small measure because of the development of in vitro systems competent to catalyze splicing and polyadenylation of simple precursor RNAs. Upon addition to a splicing extract, an exogenous precursor RNA rapidly assembles into a large ribonucleoprotein complex (the spliceosome) containing hnRNP and snRNP factors known to be required for the cleavage and ligation steps of splicing. The next few years promise to be exciting ones for the splicing field during which the rules of spliceosome assembly and participation in the splicing reaction will be determined. We plan to investigate both the assembly and activity of the spliceosome using in vitro processing extracts. Seven experimental approaches currently in use in the laboratory will be used to address basic questions about spliceosome structure and function, including: 1. Examination of snRNP protein phosphorylation and its relationship to assembly and activity of the spliceosome; 2. Development of two-dimensional gels for the examination of spliceosomes; 3. Fractionation of splicing extracts using size-based approaches; 4. Use of RNP gel electrophoresis to deduce the pathway of spliceosome assembly and disassembly; 5. Examination of the involvement of matrix proteins in in vitro splicing; 6. Examination of snRNP assembly and sub-populations by RNP gel analysis of complexes assembled on isolated snRNAs, and 7. Examination of the relationship between splicing and polyadenylation through the use of precursor RNAs containing introns and poly(A) sites.